Notes:
Summary The transport through the epithelial cell layer of the renal proximal tubule proceeds in principle by passive paracellular and active transcellular transport. The active transcellular transport is mostly secondary active. This means it proceeds coupled with the flux of Na+ ions, where-by the transcellular gradient of sodium, created by the (Na++K+)-ATPase, located at the contraluminal cell side, provides the main driving force. Once in the cell the substances leave the other cell side by a Na+-independent, but carrier-mediated transport system. Using microperfusion and electrophysiological techniques as well as brush border membrane vesicle preparation the Na+-H+ countertransport and the Na+-cotransport of amino acids, phosphate, sulfate, thiosulfate, bile acids, aliphatic-aromatic monocarboxylic acids (lactate) and dicarboxylic acids was studied. Special emphasis will be given to the bidirectional transport of thiosulfate as well as to the specificity of the monocarboxylic acid and dicarboxylic acid transport system.

Notes:
Abstract In order to study the characteristics of contraluminal phosphate transport the stopped flow microperfusion technique [13] has been applied. By measuring the time-dependent decrease of interstitial33Pi concentration at different starting concentrations a simple diffusion kinetics with a permeability coefficient of 7.5±1.0 · 10−8 cm2 s−1 was found. Such a kinetic was so far only observed with 2-deoxy-d-glucose. This substance, however, is transported in addition by facilitated diffusion as was seen by paraaminohippurate, methylsuccinate and sulfate. The contraluminal transport of phosphate was inhibited by H2-DIDS (5 mmol/l). It was, however, not influenced by omission of Na+ from the perfusates, by addition of sulfate (150 mmol/l), methylsuccinate (50 mmol/l), arsenate (50 mmol/l), the Hg-compound mersalyl (5 mmol/l), high and low phosphate diet and pH changes between 6.0 and 8.0. The data indicate that phosphate, which is reabsorbed from the lumen by a Na+-dependent transport system, leaves the cell by a rather unspecific contraluminal diffusion pathway.

Notes:
Abstract In order to evaluate whether N-containing substrates interact with the organic “anion” (p-aminohippurate, PAH) or only with the organic “cation” (N 1-methylnicotinamide, NMeN) transport system or with both, the stop-flow peritubular capillary microperfusion method was applied in the rat kidney in situ and the apparent K i values of several classes or organic substrate against contraluminal NMeN and PAH transport were determined. Organic “anion” and organic “cation” transport are in inverted commas because neither transporter sees the degree of ionization in bulk solution, and they also accept nonionizable substrates [Ullrich KJ, Rumrich G (1992) Pflügers Arch 421:286–288]. Amines must be sufficiently hydrophobic (phenylethylamine, piperidine, piperazine) in order to interact with NMeN transport. Additional Cl, Br, NO2 or other electronegative groups render them inhibitory towards PAH transport also. Such bisubstrate amines were identified as follows: metoclopramide, bromopride, diphenhydramine, bromodiphenhydramine, verapamil, citalopram, ketamine, mefloquine, ipsapirone, buspirone, trazodone, H7 and trifluoperazine. Imidazole analogues interact with both transporters if they bear sufficiently hydrophobic alkyl or aryl groups or electronegative sidegroups. Bisubstrate imidazole analogues are tinidazole, pilocarpine, clonidine, azidoclonidine and cimetidine. Pyridines and thiazoles interact with the NMeN transporter if they have an additional ring-attached NH2 group. Again with an additional Cl, Br, or NO2 group the aminopyridines and aminothiazoles also become inhibitors for the PAH transporter. Amongst the guanidines only substances with several electronegative side-groups such as guanfacine, amiloride, benzylamiloride and ranitidine, interact with both transporters. Amongst the phenylhydrazines only 4-bromophenylhydrazine interacts with the NMeN transporter and 4-nitrophenylhydrazine with both transporters. Quinoline (isoquinoline) and its amino and hydroxy analogues interact with both transporters, their pKa values correlate directly with the affinity to the NMeN transporter and reciprocally with their affinity to the PAH transporter. In experiments with labelled substrates only the sufficiently hydrophilic cimetidine, amiloride and ranitidine show a saturable transport, which can be inhibited by probenecid (apalcillin) and tetraethylammonium in an additive manner. The highly hydrophobic substrates verapamil, citalopram, imipramine, diltiazem and clonidine enter the cell very fast in an unsaturable and uninhibitable manner, apparently in the undissociated form, since N-methyl-4-phenylpyridinium, which — disregarding its ionization — is similarly hydrophobic, shows a transport behaviour similar to that of tetraethylammonium [Ullrich et al. (1991) Pflügers Arch 419:84–92]. Ethidium bromide and dimidium bromide, which have a permanent cationic quaternary nitrogen and two sufficiently electronegative NH2 groups, also interact with both transporters. The data indicate that a molecule qualifies as a bisubstrate if it carries both the essentials for organic anion (PAH) transport: hydrophobicity, sufficient acidity or electron-attracting O, OH, Cl, Br, NO2 groups, plus the essentials for organic cation transport: hydrophobicity, sufficient basicity or electron-donating N-containing groups. The nitrogen atoms in the N-containing molecules quinoline (pK a 4.9), isoquinoline (pK a 5.4) and benzylpyridine (pK a 5.13) are of such low basicity that they apparently can also interact with the PAH transporter. Apparent hydrophobicity (disregarding ionization) determines interaction with the transporters, while real hydrophobicity [log (octanol distribution values)] determines the diffusion through the lipid bilayer of the cell membrane.

Notes:
Abstract In order to test what chemical structure is required for a substrate to interact not only with the contraluminal organic anion (p-aminohippurate, PAH) transporter, but also with the organic cation (N 1-methylnicotinamide, NMeN, or tetraethylammonium, TEA) transporter, the stop-flow peritubular capillary perfusion method was applied and app. K i values were evaluated. Zwitterionic hydrophobic dipeptides not only interact with PAH but also with NMeN transport although with lower inhibitory potency (K i,PAH=0.2–1.4; K i,NMeN 614 mmol/l). Amongst the zwitterionic cephalosporins, which all inhibit PAH transport, the amino cephalosporin analogue cefadroxil was identified to interact also with NMeN transport (K i,PAH = 3.0, K i,NMeN=11.2 mmol/l). All Zwitterionic naphthyridine and oxochinoline gyrase inhibitors tested inhibit NMeN transport with app. K i,NMeN values between 1.2 mmol/l and 4.7 mmol/l; the naphthyridine analogues show a good inhibitory potency against PAH transport (K i,PAH ≈ 0.4 mmol/l), the piperazine-containing quinolone analogues have a moderate inhibitory potency (K i,PAH=1.1–2.5 mmol/l) and the piperazine-containing pipemidic acid did not inhibit PAH transport at all. Zwitterionic thiazolidine carboxylate phosphamides also interact with both transporters (app. K i,PAH ≈ 3.0; app. K i,NMeN ≈ 18.0 mmol/l). The nonionizable oxo- and hydroxy-group-containing corticosteroid hormones also interact with the two transporters. (a) An OH group in position 21 is necessary for interaction with the PAH transporter, but not for interaction with the TEA transporter. (b) Introduction of an OH group in position 17α abolishes interaction with the TEA transporter, but has different effects with the PAH transporter. (c) Introduction of an OH group in position 6 abolishes interaction with both, the PAH and the TEA transporter. (d) A change of the side-group in position 11 of corticosterone from -OH to -H to=O enhances interaction with the PAH transporter but has no effect on the interaction with the TEA transporter. Nonionizable 4- or 5-androstene analogues inhibit both transporters with app. K i between 0.16 mmol/l and 0.64 mmol/l, if the steroids are soluble in a concentration greater than 1 mmol/l. Nonionizable oxazaphosphorins with more than one chloroethyl group interact with the PAH transporter with app. K i between 0.84mmol/l and 4.9mmol/l and with the NMeN transporter with app. K i between 3.2 mmol/l and 18.7 mmol/l. Thus a substrate interacts with both transporters if it is sufficiently hydrophobic, possesses acidic and/or electron-attracting plus basic and/or electron-donating groups, or possesses several electron-attracting nonionizable groups (O, OH, Cl). A certain spatial arrangement of the interacting groups seems to be necessary.

Notes:
Abstract In order to study the specificity of the contraluminal para-aminohippurate (PAH) transport system, the inhibitory potency of monocarboxylates on the3H-PAH influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: if a homologous series of fatty acids with increasing chain length is tested, inhibition of contraluminal PAH influx is first seen with valerate (app.K i 1.4 mmol/l), increasing up to nonanoate (app.K i 0.06 mmol/l) and remaining in this range up to duodecanoate, the last compound of this series which is sufficiently water-soluble. Similarly, the inhibitory potency of aromatic monocarboxylates increases with increasing hydrophobicity. If the fatty acids are esterified, their inhibitory potency is lost. If they are transformed to the respective aldehydes their inhibitory potency is preserved at a reduced degree. Introduction of a hydrophobic methyl-, ethyl-, or propyl-group increases the inhibitory potency. A β-, but not an α-oxo-group augments the inhibitory potency of phenylpropionate analogs, an OH group diminishes it, and a NH2 group abolishes it. Among phenyl-fatty acids an increase in affinity is observed from phenyl- 〈 benzoylamine-〈 phenoxy- 〈 benzoyl-acetate and-propionate. All monocarboxylate compounds, so far tested, do not inhibit contraluminal sulfate and Na+/succinate influx. The data indicate that the PAH transporter interacts with monocarboxylates and also with aldehydes which have a hydrophobic moiety. An additional oxo-group facilitates the interaction. Thus, the benzoyl compounds show the highest affinity observed.

Notes:
Abstract In order to study contraluminal sulfate transport the influx rate of35SO 4 2− from the interstitium into cortical tubular cells has been determined. Preloading of the rat with sulfate augmented contraluminal35SO 4 2− influx; preperfusion with sulfate-free solutions diminished it. The contraluminal35SO 4 2− influx in sulfate-loaded animals followed two parameter kinetics (K m 1.4 mmol/l,J max 1.2 pmol·s−1·cm−1). The contraluminal35SO 4 2− influx (starting concentration 10 μmol/l) did not change when the K+ concentration was varied between 4 and 40 mmol/l and the Ca2+ concentration from zero to 3 mmol/l. Omission of Na+ from the perfusates augmented contraluminal35SO 4 2− influx markedly. The increase is larger at pH 6 than at pH 7.4. Changes of pH affect contraluminal35SO 4 2− influx only when the solutions are Na+- and K+-free. Under these conditions the35SO 4 2− influx decreased when the ambient pH was raised from pH 6.0 to pH 8.0. Thiosulfate, selenate, molybdate, oxalate, phosphate, arsenate, and bicarbonate exerted competitive inhibition, while formate, 2-oxoglutarate and paraaminohippurate showed a biphasic response: inhibition at 50 mmol/l, no inhibition at 150 mmol/l. Chloride and bicarbonate inhibited35SO 4 2− influx at 10 μmol/l35SO 4 2− , but augmented sulfate influx at 5 mmol/l35SO 4 2− concentration in rats not preloaded with sulfate. The data indicate the presence of a contraluminal sulfate transport system which is shared by a variety of inorganic and organic anions. The biphasic behaviour of some anions suggests parallel pathways leading to a cis-inhibition at small and trans-stimulation at high anion concentrations. Na+ and H+ may be cotransported or interact with the transport system at a modifier site.

Notes:
Abstract In order to study the specificity of the contraluminal sulfate transport system the inhibitory potency of salicylate analogs (5 mmol/l each) on the35SO 4 2− influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 2-hydroxybenzoate (salicylate), per se, did not inhibit contraluminal35SO 4 2− influx. The same holds when an additional NH2-group was introduced in position 4 or 5, or when an additional Cl-group was introduced in position 4. When an additional Cl- or NO2-group was introduced in position 5 a moderate inhibition was seen (app.K i≈4 mmol/l). However, introduction of 2 Cl- or 2 NO2-groups in position 3 and 5 creates compounds with strong inhibitory potency (app.K i≈0.5 mmol/l). 2-hydroxy-3,5-iodobenzoate inhibited too, but with a smaller inhibitory potency (app.K i≈2.3 mmol/l). 2-hydroxybenzoate analogs, which have a carboxy- or sulfo-group in position 5, exerted strong inhibition, those with a acetyl- or butyryl-group exerted moderate inhibition. 1-Naphthol-2-carboxylate did not inhibit, while 1-naphthol-4-sulfamoyl-2-carboxylate did. Amongst the dihydroxybenzoates, 2,3- and 2,5-dihydroxybenzoate did not inhibit contraluminal35SO 4 2− influx, while 2,4- and 2,6-dihydroxybenzoate did. The data indicate that a hydroxy-group in ortho-position and an electro-negative group in the meta-position to the carboxyl group and paraposition to the hydroxy-group are essential for interaction with the contraluminal sulfate transport system. The ability of 2,6-dihydroxybenzoate to inhibit might be explained by its ability to undergo mesomeric conformation.

Notes:
Abstract In order to study the specificity for the contraluminal sulfate transport system the inhibitory potency of disulfonates, di-, tricarboxylates and sulfocarboxylates on the35SO 4 2− influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1) Methane- and ethane-disulfonate as well as benzene-1,3-disulfonate inhibit contraluminal35SO 4 2− influx (with an (app.K i of 〈6 mmol/l), while benzene-1,2- and 1,4-disulfonate do not. 2) The inhibitory potency of 1,3-benzene disulfonate is slightly augmented by an additional NH2 − or OH-group in position 4. However, OH-groups at position 4 and 5 or 4 and 6 abolish the inhibitory potency. 3) The naphthalene disulfonates tested inhibit only if they have an OH-group in ortho-position to one SO3H group. 4) The stilbene disulfonates H2DIDS and DNDS inhibit the contraluminal35SO 4 2− influx with high (app.K i≈0.8 mmol/l), DADS with lower potency (app.K i≈6 mmol/l). 5) Amongst the tested aliphatic di- and tricarboxylates inhibition was exerted by oxalate (app.K i 1.1 mmol/l) and maleate (app.K i 3.8 mmol/l), but not by malonate, hydroxymalonate and citrate. 6) Out of the tested benzenedicarboxylates only those inhibit which have the COO−-groups directly on the ring in 1,2 and 1,3 position (app.K i 4.0 and 2.7 mmol/l), but not in the 1,4 position. An additional OH-group in position 4 augments the inhibitory potency of 1,3 benzene-dicarboxylates (app.K i 0.8 mmol/l), while an OH group on position 5 abolishes it. 7) The benzene tricarboxylates (BTC) inhibit in the sequence 1,2,3-BTC〉1,3,5-BTC〉1,2,4-BTC (app.K i 0.9, 1.5 and 4.2 mmol/l, respectively). 8) The carboxy-benzene-sulfonates inhibit also in the 1,2 and 1,3 position only (app.K i 6.7 and 5 mmol/l), but not in the 1,4 position. Addition of an −OH-group to the 3-carboxy-1-benzene-sulfonate forming 4-hydroxy-3-carboxy-1-benzene-sulfate augments the inhibitory potency drastically (app.K i 0.32 mmol/l), while a NH2 substitution at the same position leaves it unchanged (app.K i 4.7 mmol/l). If, however, ethylamine instead of NH2 is used as substituent, the inhibitory potency is almost as high as of 4-hydroxy-3-carboxy-1-benzene-sulfonate (app.K i≈0.6 mmol/l). Amongst the dicarboxy-benzene-sulfonates, 3,4-carboxy-benzene-1-sulfonate inhibits (app.K i ca. 2 mmol/l), while 3,5-carboxy-benzene-1-sulfonate does not. The data indicate that a strong interaction of substrate with the sulfate transporter is given, when two charged groups (COO− and/or SO 3 − ) are present in a distance equivalent to the meta-position on the benzene ring and an additional hydrogen bond forming OH- or −NH-group. Hydrogen bond forming groups and charged groups in other positions usually abolish the inhibitory potency.

Notes:
Abstract In order to evaluate the specificity for the contraluminal sulfate transport system the inhibitory potency of phenol- and sulfonphthaleins, of sulfamoyl-compounds (diuretics) as well as diphenylamine-2-carboxylates (Cl− channel blockers) on the35SO 4 2− influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1) Phenolsulfonphthalein (phenol-red) inhibited with an app.K i-value of 1.7 mmol/l, while analogs which had additional Br-atoms in position 3 and/or 5, i.e. bromphenol-blue, bromcresol-purple and bromcresol-green, inhibited with an apparentK i of 0.1 and 0.5 mmol/l respectively. 2) Phenolphthalein and tetrabromphenolphthalein did not inhibit, while the disulfonate dyes bromsulfalein, fuchsin acid and indigocarmine inhibited with aK i between ≈1 and 3 mmol/l. The highest inhibitory potency in this class of compounds was seen with orange G (app.K i 0.07 mmol/l). The monosulfonate dyes tested, fluoresceinsulfonate and orange I inhibited moderately with an app.K i of ≈5 mmol/l. 3) The 3-sulfamoyl compounds inhibited to a varying degree, when they had a neighbouring −NH-group (furylmethylamino-group), i.e. in position 6 to the COOH or SO3H-group, or when they had a phenoxy-group in position 4. 4) 4-sulfamoylbenzoate and the related compounds probenecid, acetazolamide and hydrochlorothiazide inhibited with an app.K i between 4 and 7 mmol/l. 5) All diphenylamine-2-carboxylate analogs inhibited with an app.K i between 3 and 5 mmol/l, even when the −NH-group was replaced by an =O-group or the benzene ring was replaced by a pyrimidine ring, but not when it was replaced by a thiophen ring. In contrast, 4-phenylaminepyridine-3-sulfonate was ineffective, while diphenylamine-2-amino sulfonate exerted the highest inhibition of this group with an app.K i of 1.4 mmol/l. When, however, the aminosulfonate group was replaced by a methylsulfonamide, the inhibitory potency disappeared. The data can be explained by inhibitory patterns found in previous papers for disulfonates [29], sulfonates with a hydrophobic moiety [28] or neighbouring OH-group [28, 29], carboxylates with a neighbouring −NH- or OH-group in position 2- and an electron-attracting group in position 5 [30].

Notes:
Abstract In order to study the specificities of the contraluminal anion transport systems, the inhibitory potency of substituted benzene analogs on influx of [3H]PAH, [14C]succinate, and [35S]sulfate from the interstitium into cortical tubular cells has been determined in situ: (1) Contraluminal [3H]PAH influx is moderately inhibited by benzene-carboxylate and benzene-sulfonate, and strongly by benzene-dicarboxylates,-disulfonates and carboxy-benzene-sulfonates, if the substituents are located at positions 1 and 3 or 1 and 4. The affinity of the PAH transporter to polysubstituted benzoates increases with increasing hydrophobicity, decreasing electron density at the carboxyl group and decreasing pKa. Similar dependencies are observed for phenols. Benzaldehydes which do not carry an ionic negative charge are accepted by the PAH-transporter, if they possess a second partially charged aldehyde or NO2-group. (2) Contraluminal [14C]succinate influx is inhibited by benzene 1,3- or 1,4-dicarboxylates,-disulfonates and 1,3-or 1,4-carboxybenzene-sulfonates. Monosubstituted benzoates do not interact with the dicarboxylate transporter, but NO2-polysubstituted benzoates do. Phenol itself and 2-substituted phenol interact weakly possibly due to oligomer formation. (3) The contraluminal sulfate transporter interacts only with compounds which show a negative group accumulation such as 3,5-dinitro- or 3,5-dichloro-substituted salicylates. The data are consistent with three separate anion transport systems in the contraluminal membrane: The PAH transporter interacts with hydrophobic molecules carrying one or two negative charges (−COO−, −SO 3 − ) or two or more than two partial negative charges (−OH, −CHO, −SO2NH2, −NO2). The dicarboxylate transporter requires two electronegative ionic charges (−COO−, −SO 3 − ) at 5–9 Å distance or one ionic and several partial charges (−Cl, −NO2) at a favourable distance. The sulfate transporter interacts with molecules which have neighbouring electronegative charge accumulation.